Cell traces and animal fashions
Rat cardiomyocytes (H9c2), human umbilical vein endothelial cells (HUVECs), and human mesenchymal stem cells (hMSCs) have been obtained from the American Sort Tradition Assortment (ATCC). All cell traces have been cultured and maintained following ATCC’s normal protocols. Male Sprague-Dawley (SD) rats (6–8 weeks outdated, weighing 200–250 g) and male C57BL/6 mice (6–8 weeks outdated, weighing 18–22 g) have been bought from the Southern Medical College Experimental Animal Heart. Animals have been housed in a selected pathogen-free (SPF) setting underneath managed situations of twenty-two ± 2 °C, 50–60% relative humidity, and a 12 h gentle/darkish cycle, with free entry to meals and water.
Pathological fashions of cells and animals and remedy
An I/R mannequin was developed in H9c2 cardiomyocytes (ATCC, CRL-1446). The tradition medium was eliminated, and cells have been washed twice with glucose-free DMEM (Gibco, C11995500BT). Glucose-free, serum-free DMEM was then added, and the cells have been incubated in a hypoxic chamber at 37 °C underneath 5% CO₂ and 95% N₂ for six h to induce hypoxia. After finishing the hypoxia and glucose deprivation section, the medium was changed with high-glucose DMEM supplemented with 10% FBS. Exo-WT and Exo-I-S have been added at a dosage of three,000 particles per H9c2 cell (with an exosome protein focus of 20 µg/mL), together with PBS as a management. The cells have been subsequently cultured underneath normal situations at 37 °C with 5% CO₂ for 12 h.
For the in vivo experiment was authorised by Nan-fang hospital, southern medical college (IACUC-LAC-20240822-005), animal care and remedy have been carried out following institutional procedures and nationwide legal guidelines and rules, male SD rats have been anesthetized with isoflurane, and a thoracotomy was carried out on the fourth intercostal area to reveal the guts. A 6 − 0 polypropylene suture was positioned 2–3 mm under the origin of the left anterior descending artery, between the left atrial appendage and the arterial cone, to induce myocardial ischemia/reperfusion damage. After 1 h of ischemia, the suture was eliminated to permit reperfusion. Intramyocardial injections of Exo-WT and Exo-I-S options (dose: 1 × 10¹¹ particles/kg physique weight; exosome protein focus: 20 µg/mL) or an equal quantity of PBS have been administered into the infarct space (recognized by pale discoloration after ligation).
Development of MSCIns cells
A dual-gene expression plasmid, pIRES-SIRT3/GPI + Insulin, was constructed utilizing molecular cloning methods. Human SIRT3 and insulin genes have been amplified by PCR, with a GPI-anchoring sequence added to the three’ finish of the insulin gene. The amplified merchandise have been then cloned into MCS A and MCS B websites of the pIRES bicistronic expression vector. After profitable sequencing verification, plasmid DNA was extracted and transfected into MSCs utilizing Lipofectamine™ 2000 (ThermoFisher, 11668019) to generate SIRT2/GPI + Insulin expressed MSC (MSCIns). Transfected cells have been harvested 12 and 24 h post-transfection for immunofluorescence and Western blot validation.
Preparation of Exo-I-S and Exo-WT
Each transfected and non-transfected MSCs (ATCC, PCS-500-012) have been initially cultured for 48 h. The medium was then changed with serum-free DMEM for a further 24 h. The tradition supernatant was collected and sequentially centrifuged for exosome isolation: 300 × g for 10 min, 2,000 × g for 10 min, and 100,000 × g for 70 min utilizing an Optima MAX-XP ultracentrifuge (Beckman, USA). The supernatant was discarded, and the pellet was washed with PBS, adopted by a last ultracentrifugation step at 100,000 × g for 70 min. Purified exosomes have been saved at − 80 °C till evaluation.
Characterization of Exo-I-S and Exo-WT
Exosome particle dimension evaluation
Nanoparticle monitoring evaluation (NTA) was carried out utilizing the ZetaView PMX 110 instrument (Particle Metrix) and its accompanying software program (ZetaView 8.02.28). Exosomes have been diluted appropriately in particle-free PBS earlier than being loaded into the pattern chamber. The dimensions distribution and focus of exosomes have been decided at a wavelength of 405 nm. Particle dimension knowledge have been quantified and recorded.
Protein quantification
Protein focus was decided utilizing a BCA Protein Assay Equipment (Beyotime, P0012), following the producer’s protocol. Samples and normal proteins (BSA) have been added to a 96-well plate, blended with BCA working reagent, and incubated at 37 °C for 30 min. Absorbance was measured at 562 nm in microplate reader, and protein focus was calculated primarily based on a normal curve (0–2,000 µg/mL).
Transmission electron microscopy
Exosome samples have been dropped onto carbon-coated copper grids, air-dried at room temperature, and negatively stained with 2% phosphotungstic acid for five min. Extra stain was eliminated with filter paper, and the grids have been examined underneath a transmission electron microscope (TEM) (JEM-1230, JEOL) to evaluate the morphology and dimension of the exosomes.
Western blot
Complete protein was extracted from cells and exosomes and separated on 10–15% SDS-polyacrylamide gels, primarily based on the molecular weight of the goal proteins. Proteins have been transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, IPVH00010) and blocked with 5% skim milk for 1 h. Membranes have been incubated in a single day at 4 °C with major antibodies towards: SIRT3 (1:1500, ABclonal, A5419), Insulin (1:2000, HUABIO, EM80714), CD63 (1:1500, Abcam, ab217345), CD9 (1:1800, Abcam, ab2215), ALIX (1:1500, Abcam, ab186429), Calnexin (1:1500, Abcam, ab22595), GAPDH (1:20000, Abcam, ab181602), AKT (1:1000, Abcam, ab179463), p-AKT (1:1000, Abcam, ab38449), PI3K (1:1000, Abcam, ab86714), p-PI3K (1:800, Abcam, ab182651), and Glut4 (1:1500, ABclonal, A25174). Secondary antibodies similar to goat anti-mouse IgG (1:3000, Abcam, ab205719) and goat anti-rabbit IgG (1:3000, Abcam, ab205718) have been utilized. Protein bands have been visualized utilizing enhanced chemiluminescence (ECL) reagents (Beyotime, P0018S) and imaged utilizing a ChemiDoc MP system (Bio-Rad).
Exosome uptake assay
The uptake of exosomes by H9c2 cardiomyocytes and HUVEC endothelial cells was monitored utilizing PKH26-labeled (200 µg/mL, Sigma, MINI26) management Exo (Exo-WT) and Exo-I-S. Labeled exosomes have been added to the H9c2 tradition medium, and cells have been incubated for 3, 6, 12, 24, and 36 h. After incubation, cells have been washed with DPBS and glued with 4% paraformaldehyde (PFA). Cell nuclei have been stained with 4’,6-Diamidino-2-phenylindole (DAPI, Invitrogen) for visualization. Fluorescence imaging was carried out utilizing a confocal laser scanning microscope (LSM 880, Zeiss). Pictures of three impartial samples have been captured for every group, and ImageJ was utilized to quantify the relative fluorescence depth utilizing the automated threshold methodology to pick the fluorescent areas.
GPI-Insulin molecular modeling and Docking prediction
The three-dimensional construction of the GPI-Insulin fusion protein was predicted utilizing AlphaFold 3.0. By inputting the amino acid sequence, AlphaFold 3.0 generated a 3D structural mannequin and offered the expected Template Modeling Rating (pTM). The expected construction was then imported into AutoDock Vina for molecular docking evaluation with the insulin receptor (IR). The construction of the insulin receptor was obtained from the Protein Information Financial institution (PDB). Previous to docking, preprocessing was carried out utilizing AutoDock Instruments, which included eradicating water molecules, including polar hydrogen atoms, calculating Gasteiger costs, and saving the recordsdata in PDBQT format. Through the docking course of, a grid field was set to cowl the receptor’s lively website, and the Lamarckian genetic algorithm was employed to finish the docking. The binding mode and binding power of the GPI-Insulin fusion protein with the insulin receptor have been subsequently decided.
Biocompatibility analysis
Tube formation assay
HUVECs (ATCC, CRL-1730) have been seeded into 96-well plates pre-coated with 10 mg/mL Matrigel matrix (Corning, 354234) to simulate an in vitro angiogenesis setting. After seeding at an acceptable density, tradition medium containing exosomes (e.g., Exo-I-S, Exo-WT, or management) was added. After 6–12 h of incubation, the formation of tubular buildings was noticed and imaged utilizing an inverted microscope (Leica, DMi8). Tube size and the variety of department factors have been analyzed utilizing ImageJ software program to guage the consequences of various remedies on angiogenesis.
Dwell/Lifeless cell assay
H9c2 cells at acceptable confluency have been handled with tradition medium containing Exo-I-S, Exo-WT, or PBS for twenty-four h. After eradicating the medium and washing the cells with PBS, a dye working resolution was ready in response to the producer’s directions: Calcein-AM (2 µM, Invitrogen, C3099) and PI (5 µg/mL, Invitrogen, P1304MP). Cells have been incubated at 37 °C for 15–30 min, and fluorescence pictures have been captured utilizing a fluorescence microscope.
Organ toxicity evaluation
Sterile Exo-I-S, Exo-WT, or PBS options have been ready and administered to C57BL/6 mice through tail vein injection at a quantity of 10 mL/kg physique weight. The management group obtained an equal quantity of PBS. Seven days after administration, mice have been sacrificed underneath extreme anesthesia, and main organs have been quickly harvested and glued in 4% paraformaldehyde. Hematoxylin and eosin (H&E) staining was carried out to evaluate histopathological adjustments.
Immunofluorescence stain
Cells have been permeabilized with 0.1% Triton X-100 at room temperature for 10 min, adopted by blocking with 1% BSA/PBST buffer for 1 h. The cells have been incubated in a single day at 4 °C with the first antibody Glut4 (1:200, ABclonal, A25174), SIRT3 (1:200, ABclonal, A5419), Insulin (1:200, Abcam, ab46707). After washing with PBS, cells have been incubated with Anti-rabbit Alexa Fluor 488-conjugated secondary antibody (1:1000, Cell Signaling, 4412) Anti-mouse Alexa Fluor 594-conjugated secondary antibody (1:1000, Cell Signaling, 8890) and at nighttime for two h. Nuclei have been stained with DAPI (1 µg/mL, 1:1000, Sigma-Aldrich, D9542).
Mitochondrial morphology evaluation
Mitochondrial Electron microscopy
Cells have been washed with serum-free medium and glued with 4% glutaraldehyde (Sigma-Aldrich, 814393) and 4% paraformaldehyde (Sigma- Aldrich, 818715). Samples have been dehydrated in a graded ethanol collection and embedded in LX-812 resin (Ladd Analysis Industries Inc.). Ultrathin sections have been stained with uranyl acetate for 30 min and lead citrate for 10 min, and noticed utilizing a FEI Tecnai G12 Spirit BioTwin transmission electron microscope (FEI Firm, Hillsboro) at an acceleration voltage of 120 kV.
JC-1 staining
To judge mitochondrial membrane potential underneath totally different Exo remedies, JC-1 staining was carried out utilizing the MitoProbe JC-1 Assay Equipment (Invitrogen, M34152). Cells have been incubated with JC-1 (10 µL, 200 µM) for 30 min, and fluorescence was detected at 488 nm and 633 nm utilizing a confocal laser scanning microscope (Zeiss LSM 880).
Mito-Tracker evaluation
A 1 mM inventory resolution of Mito-Tracker Pink was ready in anhydrous DMSO and saved at − 20 °C at nighttime. After eradicating the tradition medium, pre-warmed Mito-Tracker Pink CMXRos staining resolution (Beyotime, C1999S) was added to the cells, which have been incubated at 37 °C for 30 min. Following incubation, the staining resolution was changed with recent medium, and fluorescence was noticed utilizing a confocal laser scanning microscope (Zeiss LSM 880).
Mitochondrial oxidative stress detection
ROS measurement
Reactive oxygen species (ROS) have been measured utilizing the DCFH-DA probe (ab113851). The DCFH-DA resolution was diluted 1:1000 with serum-free medium and saved shielded from gentle. Cells have been washed twice, then incubated with 100 µL of the diluted DCFH-DA resolution at 37 °C for 30 min. After incubation, cells from the oxidative stress mannequin have been washed 3 times with serum-free medium to take away extra probe not taken up by cells, adopted by addition of recent medium and additional incubation for 30 min. Cells have been then collected by centrifugation, resuspended within the diluted DCFH-DA resolution, and analyzed by circulate cytometry. A complete of 10,000 cells per pattern have been measured for fluorescence depth, and the common fluorescence depth was calculated. Outcomes have been expressed because the ratio of fluorescence depth between experimental and regular teams.
SOD1 and SOD2 measurement
SOD1 and SOD2 ranges have been measured utilizing ELISA kits (CBIBO BIO, CB11409-Ra and CB10619-Ra). Cells have been washed 3 times with PBS and centrifuged (3000 rpm, 5 min, 4 °C). After including phosphate-buffered saline and extraction buffer (1:1000), cells have been ultrasonicated (3 s bursts, 10 s intervals, 30 cycles) and centrifuged once more (3000 rpm, 20 min, 4 °C). Supernatants have been collected and stored on ice. Requirements (50 µL) and samples (40 µL dilution buffer with 10 µL pattern, 5-fold diluted) have been added to wells. Aside from blanks, 100 µL enzyme conjugate was added; plates have been sealed and incubated at 37 °C for 60 min. Wells have been washed 5 instances with 20-fold diluted wash buffer, then incubated with 50 µL every of substrate A and B for 15 min at 37 °C at nighttime. The response was stopped with 50 µL cease resolution, turning coloration from blue to yellow. Absorbance was learn at 450 nm inside 15 min, and outcomes have been expressed as experimental-to-normal group ratios.
Apoptosis detection
Cell apoptosis was analyzed utilizing Annexin V-FITC/PI twin staining. Cells have been collected by trypsinization, washed twice with PBS, and resuspended in 1× Annexin V binding buffer (BD Biosciences, 556547). Annexin V-FITC (5 µL) and PI dye (5 µL) have been added, and the samples have been incubated at nighttime for 15 min at room temperature. Fluorescence alerts have been detected utilizing a circulate cytometer (BD FACSCanto II).
Cell metabolic exercise evaluation
Mobile oxygen consumption charge (OCR) was measured utilizing the Seahorse XFe24 extracellular flux analyzer (Agilent, Seahorse XFe24) and the Mito Stress Check Equipment (Agilent, 103015-100). H9c2 cells have been seeded onto tradition plates (Agilent, 100882-004) and subjected to I/R remedy. On the day of measurement, cells have been washed with XFe24 medium and incubated in a CO₂-free incubator at 37 °C for two h to equilibrate. OCR was measured earlier than and after the sequential addition of glucose (10 mmol/L), oligomycin (1 µmol/L), FCCP (4 µmol/L), and rotenone/antimycin A (0.5 µmol/L). Basal respiration, ATP manufacturing, and maximal respiration have been calculated.
Mitochondrial enzymatic detection
For the actions of glycolysis enzymes, We used the 6-phosphofructokinase (PFK-1) exercise package (Solarbio, BC0530), lactate dehydrogenase (LDH) exercise package (Solarbio, BC0685), lactate content material package (Solarbio, BC2230), pyruvate dehydrogenase (PDH) exercise package (Abcam, ab287837), and ATP content material package (Solarbio, BC0300). and RT-qPCR was carried out to evaluate the enzymes within the tricarboxylic acid (TCA) cycle, particularly citrate synthase (CS) and isocitrate dehydrogenase (IDH), in addition to parts of the oxidative phosphorylation advanced, specifically NADH dehydrogenase 1-β3 (NDUFB3) and succinate dehydrogenase advanced subunit B (SDHB).The response combine included 5 µL of SsoFast Eva Inexperienced Supermix, 0.5 µL of every upstream and downstream primer (10 µM), 2 µL of cDNA template, and a pair of µL of ultrapure water, totaling 10 µL. We used β-tubulin because the reference gene, and the relative expression ranges of the goal genes have been calculated utilizing the 2-ΔΔCT methodology. Primers sourced from Shanghai Shenggong have been as follows: (See Desk 1).
Serum detection of CK-MB, CTnT, and LDH
Blood samples have been collected from the interior canthus vein 12 h after myocardial infarction modeling, and serum was separated by centrifugation at 3000 rpm for 10 min. The degrees of CK-MB, CTnT, and LDH have been measured utilizing enzyme-linked immunosorbent assay (ELISA) kits. The next particular kits have been used: CK-MB (Abcam, ab285275), CTnT (Abcam, ab246529), and LDH (Sigma-Aldrich, MAK066). Following the directions of the kits, serum samples have been added to the enzyme-linked plate, incubated, after which the enzyme conjugate and substrate have been added for coloration improvement. After terminating the response, the absorbance was measured at 450 nm. The concentrations of every biomarker have been calculated primarily based on the usual curve to evaluate the extent of myocardial damage.
Electrocardiogram
Previous to the experiment and 12 h after myocardial infarction modeling, rats have been anesthetized (with intraperitoneal injection of pentobarbital sodium at a dose of 40 mg/kg) and glued on the experimental platform. Their limbs have been uncovered, and electrocardiogram electrodes have been related (one for every forelimb and hindlimb). Care was taken to make sure that the electrodes had good contact with the pores and skin. An animal-specific electrocardiogram system (ADInstruments, ML136) was used to file.
Echocardiography
Utilizing the Vevo high-resolution small animal ultrasound imaging system and a high-frequency transducer, cardiac ultrasound examinations have been carried out via the sternum on the 14th day post-surgery. SD rats have been anesthetized whereas sustaining spontaneous respiration, and two-dimensional pictures of the left ventricle have been obtained within the short-axis view on the degree of the left ventricular papillary muscle tissues. Key measurements included coronary heart charge, left ventricular systolic wall thickness (Awsth; Awdth), diastolic wall thickness (Pwsth; Pwdth), left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular end-diastolic exterior diameter (EXLVDd), and left ventricular short-axis fractional shortening (FS%). The top-diastolic quantity (EDV) and end-systolic quantity (ESV) have been then calculated utilizing built-in correction formulation.
FS was calculated utilizing the method: FS = [(LVEDd – LVESd) / LVEDd] × 100%, and ejection fraction (EF) was decided utilizing the method: EF = (EDV – ESV) × 100% / EDV.
Histological evaluation
H&E staining
Coronary heart tissue Sect. (5 μm thick) have been ready from paraffin-embedded blocks. Sections have been deparaffinized, stained with hematoxylin (Beyotime, C0105S), differentiated in acidic water, and counterstained with eosin. After dehydration with ethanol and clearing with xylene, the slides have been mounted and analyzed underneath a microscope.
WGA staining
Mounted tissue samples have been incubated in PBS containing WGA fluorescent dye at room temperature for two h, adopted by washing 3 times with PBS. Nuclei have been counterstained with DAPI for 30 min, and the slides have been washed once more earlier than imaging.
Sirius crimson staining
Mounted tissue samples in 4% paraformaldehyde have been dehydrated, embedded in paraffin, and sectioned. After deparaffinization and rehydration, the sections have been stained with Sirius Pink resolution (Sigma-Aldrich, 365548) for 1 h. Following staining, the sections have been dehydrated with absolute ethanol, cleared with xylene, and mounted with a coverslip for microscopic evaluation.
Tissue Immunofluorescence
Tissue sections have been baked at 70 °C for 30 min utilizing an in situ hybridization instrument. The processed sections have been rapidly immersed in xylene twice, for five min every, for deparaffinization. Gradient rehydration was carried out by immersing the sections for five min every in 100%, 90%, 80%, and 70% ethanol, adopted by distilled water. Endogenous peroxidase exercise was blocked, and antigen retrieval was carried out as required. Sections have been blocked with 1% BSA at room temperature for two h, then incubated with a major antibody at 37 °C for two h or in a single day at 4 °C. The next major antibodies have been utilized: SOD2(1:500, abcam, ab13534), cTnl (1:500, abcam, ab47002), CD31(1:500, Abcam, ab9498), and α-SMA (1:500, CST, 19245T), Glut4 (1:1500, ABclonal, A25174).After washing with PBS, the sections have been incubated with a fluorescently labeled secondary antibody at room temperature at nighttime for 1 h. Finally, the cells have been stained with DAPI for five min. After one other wash with PBS, an acceptable quantity of a fluorescence quenching agent was added to cowl the slides for 90 min. Following one other PBS wash, an anti-quenching mounting medium (G1221-5ML, Servicebio) was utilized to mount the sections for subsequent remark and evaluation.
Quantification of Immunofluorescence
For the fluorescent staining of SOD2 and ROS, we chosen tissue surrounding the infarct space for picture acquisition. For the fluorescent staining of CTnI, αSMA, and CD31, pictures have been acquired instantly from the infarct area. The collected pictures have been analyzed for fluorescence depth utilizing ImageJ. After splitting the fluorescence channels equivalent to the goal proteins, an automated threshold segmentation was utilized to pick the suitable highlighted sign areas. The typical fluorescence depth of those areas was then calculated, and the info have been exported.
Co-localization evaluation of Glut4 and WGA was carried out primarily based on the overlap of the crimson and inexperienced channels. ImageJ was used for co-localization evaluation following these steps: splitting the fluorescence channels for Glut4 and WGA, aligning them, subtracting background, and acquiring the grayscale values of the co-localized areas.The extent of vascular progress in tissues was assessed by evaluating immunofluorescence staining of CD31 and αSMA in tissues from every experimental group. Fluorescence channels have been separated in ImageJ, and the fluorescence threshold was adjusted to an acceptable vary. The CD31 and αSMA channel pictures have been then stacked individually. Utilizing the segmented line software, traces alongside CD31-positive vessels have been recorded, and the constructive space ratios have been calculated by evaluating them with these of the traditional group.
Statistical evaluation
All knowledge are offered as imply ± normal deviation (SD). Statistical evaluation was carried out utilizing SPSS software program (model 27.0) and GraphPad Prism software program (model 9.5). For comparisons amongst a number of teams, one-way evaluation of variance (ANOVA) was employed, offered that the info met the assumptions of normality and homogeneity of variance. A significance degree of p < 0.05 was set. For knowledge that didn’t fulfill these assumptions, non-parametric exams such because the Kruskal-Wallis check have been used for evaluation. Moreover, for comparisons between two impartial teams, a two-sample t-test was carried out, assuming regular distribution and equal variances.